Inhibitors of nonenzymatic cross-linking

ABSTRACT

The present invention relates to compositions and methods for inhibiting nonenzymatic cross-linking (protein aging). Accordingly, a composition is disclosed which comprises an agent capable of inhibiting the formation of advanced glycosylation endproducts of target proteins by reacting with the carbonyl moiety of the early glycosylation product of such target proteins formed by their initial glycosylation. Suitable agents contain an active nitrogen-containing group, such as a hydrazine group. Particular agents comprise aminoguanidine derivatives. The method comprises contacting the target protein with the composition. Both industrial and therapeutic applications for the invention are envisioned, as food spoilage and animal protein aging can be treated.

This invention was made with partial assistance from grants from theNational Institutes of Health and the Brookdale Foundation.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of copendingApplication Ser. No. 798,032, filed Nov. 14, 1985 now U.S. Pat. No.4,758,583 which is a continuation-in-part of copending Application Ser.No. 590,820, now U.S. Pat. No. 4,665,192 filed Mar. 19, 1984, by AnthonyCerami.

RELATED PUBLICATIONS

The Applicants are co-authors of the following articles directed to thesubject matter of the present invention: "COVALENT ATTACHMENT OF SOLUBLEPROTEINS BY NONENZYMATICALLY GLYCOSYLATED COLLAGEN: ROLE IN THE IN SITUFORMATION OF IMMUNE COMPLEXES", Brownlee M., Pongor S., Cerami A.,(1983), J. Exp. Med., 158, pp. 1730-1744; and "AGING OF PROTEINS:ISOLATION AND IDENTIFICATION OF FLUORESCENT CHROMOPHORE FROM THEREACTION OF POLYPEPTIDES WITH GLUCOSE", Pongor, et al, Proc. Natl. Acad.Sci. USA, 81, pp. 2684-2688, (May, 1984), both of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to the reaction that occursbetween glucose and proteins, and more particularly to the inhibition byvarious aminoguanidine derivatives of the reaction of nonenzymaticallyglycosylated proteins leading to advanced glycosylation end products.

The reaction between glucose and proteins has been known for some time.Its earliest manifestation was in the appearance of brown pigmentsduring the cooking of food, which was identified by Maillard in 1912,who observed that glucose or other reducing sugars react with aminoacids to form adducts that undergo a series of dehydrations andrearrangements to form stable brown pigments. Maillard, C. R. Acad.Sci., 154, pp. 66-68, (1912).

In the years that followed the initial discovery by Maillard, foodchemists studied the hypothesized reaction in detail and determined thatstored and heat treated foods undergo nonenzymatic browing as a resultof the reaction between glucose and the polypeptide chain, and that theproteins are resultingly cross-linked and correspondingly exhibitdecreased bioavailability. Finot, P. A. (1982) in Modification ofProteins, eds, Feeney, R. E. and Whitaker, J. R., American ChemicalSociety, 198, pp. 91-124, Washington, D.C. At this point, it wasdetermined that the pigments responsible for the development of thebrown color that develops as a result of protein glycosylation possessedcharacteristic spectra and fluorescent properties. However, the chemicalstructure of the pigments had not been specifically elucidated.

The reaction between reducing sugars and food proteins discussed abovewas found in recent years to have its parallel in vivo. Thus, thenonenzymatic reaction between glucose and the free amino groups onproteins to yield the Amadori product, has been shown to occur withhemoglobin, wherein a rearrangment of the amino terminal of thebeta-chain of hemoglobin by reaction with glucose, forms the adductknown as hemoglobin A_(1c). The reaction has also been found to occurwith a variety of other body proteins, such as lens crystallins,collagen and nerve proteins. See, Bunn et al., Biochem. Biophys. Res.Comm., 67, pp. 103-109 (1975); Koenig et al, J. Biol. Chem., 252, pp.2992-2997 (1977); Monnier, and Cerami, A., in Maillard Reaction in Foodand Nutrition, ed. Waller, G. A., American Chemical Society, 215, pp.431-448 (1983); and Monnier and Cerami, Clinics in Endocrinology andMetabolism, 11, pp. 431-452 (1982). Moreover, brown pigments withspectral and fluorescent properties similar to those of late-stageMaillard products have also been observed in vivo in association withseveral long-lived proteins, such as lens proteins and collagen fromaged individuals. An age related linear increase in pigment was observedin human dura collagen between the ages of 20 to 90 years. See, Monnier,and Cerami, Science, 211, pp. 491-493 (1981); Monnier, and Cerami,Biochem. Biophys. Acta, 760, pp. 97-103 (1983); and, Monnier et al.,"Accelerated Age-Related Browning of Human Collagen in DiabetesMellitus", Proc. Nat. Acad. Sci., 81, pp. 583-587 (1984). Interestingly,the aging of collagen can be mimicked in vitro by the cross-linkinginduced by glucose; and the capture of other proteins and the formationof adducts by collagen, also noted, is theorized to occur by across-linking reaction, and is believed to account for the observedaccumulation of albumin and antibodies in kidney basement membrane. See,Brownlee et al, J. Exp. Med., 158, pp. 1739-1744 (1983); and Kohn et al,Diabetes, 33, No. 1, pp. 57-59 (1984).

2-Furoyl-4(5)-2(furanyl)-1H-imidazole has been isolated from the acidhydrolysates of browned proteins and is believed to be a cross-linkerfrom the nonenzymatic browning of proteins, Pongor et al., Proc. Natl.Acad. Sci. USA, 81, 2684 (1984), and U.S. Ser. No. 590,820, filed Mar.19, 1984 and entitled "Methods and Agents for Measuring Protein Aging"and U.S. Ser. No. 885,967, filed July 15, 1986 and entitled "Methods andAgents for Measuring Protein Aging".

Methods of inhibiting the Maillard reaction in vivo using aminoguanidineare known, Brownlee et al., Science, 232, 1629 (1986), and U.S. Ser. No.798,032, filed Nov. 14, 1985, and entitled "Methods and Agents forInhibiting Protein Aging". In the food industry, sulfites were foundyears ago to inhibit the Maillard reaction and are commonly used inprocessed and stored foods. Recently, however, sulfites in food havebeen implicated in severe and even fatal reactions in asthmatics. As aconsequence, the sulfite treatment of fresh fruits and vegetables hasbeen banned. The mechanism for the allergic reaction is not known. Aneed thus exists for a suitable agent for the inhibition of nonenzymaticbrowning for use in processed and stored foods, as well as in variousother pharmaceutical and diagnostic products.

Additionally, the discovery of a suitable agent for the inhibition ofnonenzymatic cross-linking would provide a means of reducing orobviating the effects of protein aging, especially in such diseasestates as diabetes mellitus, etc.

SUMMARY OF THE INVENTION

In accordance with the present invention, a method and associated agentsare disclosed for the inhibition of nonenzymatic cross-linking (proteinaging). In particular, agents for inhibiting nonenzymatic cross-linking(protein aging) due to the formation of advanced glycosylation endproducts may be selected from those materials capable of reacting withthe early glycosylation product from the reaction of glucose withproteins and preventing further reactions.

The compounds have the following structural formula: ##STR1## wherein R₁is hydrogen or a lower alkyl group of 1-6 carbon atoms, a hydroxyethylgroup, or together with R₂ may be a lower alkylene bridge of 2-4 carbonatoms; R₂ is hydrogen or a lower alkyl group of 1-6 carbon atoms ortogether with R₁ or R₃ is a lower alkylene bridge of 2-4 carbon atoms,amino, hydroxy, or an aminoalkylene group of the formula ##STR2##wherein n is an integer of 2-7 and R₆ and R₇ are independently a loweralkyl group of 1-6 carbon atoms or together with the nitrogen atom are amorpholino or piperidino group; R₃ is hydrogen, a lower alkyl group of1-6 carbon atoms, or together with R₂ or R₄ is a lower alkylene bridgeof 2-4 carbon atoms; R₄ is hydrogen, a lower alkyl group of 1-6 carbonatoms or together with R₃ is a lower alkylene bridge of 2-4 carbonatoms; or an amino group; R₅ is hydrogen, or a lower alkyl group of 1-6carbon atoms; with the proviso that at least one of R₁, R₂, R₃, R₄ or R₅is other than hydrogen; and their pharmaceutically acceptable acidaddition salts.

The lower alkyl groups referred to above contain 1-6 carbon atoms andinclude methyl, ethyl, propyl, butyl, pentyl, hexyl, and thecorresponding branched chain isomers thereof.

Thus, the compounds of this invention are substituted aminoguanidinederivatives.

Certain of the aminoguanidine derivatives useful in the method of thepresent invention are novel compounds. Correspondingly, the presentinvention relates to these novel compounds, as well as to their methods.These novel compounds are represented by the formula ##STR3## whereinR₈, R₉ and R₁₀ are hydrogen or a lower alkyl group and m is an integerof 2-4 and their pharmaceutically acceptable acid addition salts.Specifically preferred compounds are those wherein R₈, R₉ and R₁₀ areboth hydrogen and those wherein m=2.

The compounds of this invention appear to react with the glycosylationproduct thereby preventing the same from later forming the advancedglycosylation end products which lead to protein crosslinks, andthereby, to protein aging.

The present invention also relates to a method for inhibiting proteinaging by contacting the initially glycosylated protein at the stage ofthe early glycosylation product with a quantity of one or more of theagents of the present invention. In the instance where the presentmethod has industrial application, one or more of the agents may beapplied to the proteins in question, either by introduction into amixture of the same in the instance of a protein extract, or byapplication or introduction into foodstuffs containing the protein orproteins, all to prevent premature aging and spoilage of the particularfoodstuffs.

In the instance where the present method has therapeutic application,the animal host intended for treatment may have administered to it aquantity of one or more of the agents, in a suitable pharmaceuticalform. Administration may be accomplished by known techniques, such asoral, topical and parenteral techniques such as intradermalsubcutaneous, intravenous or intraperitoneal injection, as well as byother conventional means. Administration of the agents may take placeover an extended period of time at a dosage level of, for example, up toabout 25 mg/kg.

The ability to inhibit the formation of advanced glycosylation endproducts carries with it significant implications in all applicationswhere protein aging is a serious detriment. Thus, in the area of foodtechnology, economic and social benefit by making certain foods ofmarginal stability less perishable and therefore more available forconsumers. Spoilage would be reduced as would the expense of inspection,removal, and replacement, and the extended availability of the foodscould aid in stabilizing their price in the marketplace. Similarly, inother industrial applications where the perishability of proteins is aproblem, the admixture of the agents of the present invention incompositions containing such proteins would facilitate the extendeduseful life of the same. Presently used food preservatives anddiscoloration preventatives such as sulfor dioxide, known to causetoxicity including allergy and asthma in animals, might be replaced withcompounds such as those described herein.

The present method has particular therapeutic application as theMaillard process acutely affects several of the significant proteinmasses in the body, among them collagen, elastin, lens proteins, and thekidney glomerular basement membranes. These proteins deteriorate bothwith age (hence the application of the term "protein aging") and as oneof the sequelae of diabetes.

Consequently, the ability of either retard or substantially inhibit theformation of advanced glycosylation end products carries the promise oftreatment for diabetes and of course, improving the quality and perhapsduration of animal life.

Accordingly, it is a principal object of the present invention toprovide a method for inhibiting the extensive cross-linking of proteinsthat occurs as an ultimate consequence of the reaction of the proteinswith glucose, by correspondingly inhibiting the formation of advancedglycosylation end products.

It is a further object of the present invention to provide a method asaforesaid which is characterized by a reaction with an initiallyglycosylated protein identified as early glycosylation products.

It is a further object of the present invention to provide a method asaforesaid which prevents the rearrangement and cross-linking of the saidearly glycosylation products to form the said advanced glycosylation endproducts.

It is a yet further object of the present invention to provide agentscapable of participating in the reaction with the said earlyglycosylation products in the method as aforesaid.

It is a still further object of the present invention to providetherapeutic methods of treating the adverse consequences of proteinaging, manifest in the embrittlement of animal protein and the browningand spoilage of foodstuffs.

It is a still further object of this invention to provide therapeuticmethods which only minimally affect the mammalian enzyme diamineoxidase.

Other objects and advantages will become apparent to those skilled inthe art from a consideration of the ensuing description which proceedswith reference to the following illustrative drawings.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In accordance with the present invention, compositions and associatedmethods have been developed which are believed to inhibit the formationof advanced glycosylation end products in a number of target proteinsexisting in both animals and plant material. In particular, theinvention relates to a composition which may contain one or moreaminoguanidine derivatives of the formula ##STR4## wherein R₁ ishydrogen or a lower alkyl group of 1-6 carbon atoms, a hydroxyethylgroup, or together with R₂ may be a lower alkylene bridge of 2-4 carbonatoms; R₂ is hydrogen or a lower alkyl group of 1-6 carbon atoms ortogether with R₁ or R₃ is a lower alkylene bridge of 2-4 carbon atoms,amino, hydroxy, or an aminoalkylene group of the formula ##STR5##wherein n is an integer of 2-7 and R₆ and R₇ are independently a loweralkyl group of 1-6 carbon atoms or together with the nitrogen atom are amorpholino or piperidino group; R₃ is hydrogen, a lower alkyl group of1-6 carbon atoms, or together with R₂ or R₄ is a lower alkylene bridgeof 2-4 carbon atoms; R₄ is hydrogen, a lower alkyl group of 1-6 carbonatoms or together with R₃ is a lower alkylene bridge of 2-4 carbonatoms; or an amino group; R₅ is hydrogen, or a lower alkyl group of 1-6carbon atoms; with the proviso that at least one of R₁, R₂, R₃, R₄ or R₅is other than hydrogen; and their pharmaceutically acceptable acidaddition salts.

The lower alkyl groups referred to above contain 1-6 carbon atoms andinclude methyl, ethyl, propyl, butyl, pentyl, hexyl and the correspondigbranched chain isomers thereof.

The compounds are capable of inhibiting the formation of advancedglycosylation end products on such target proteins, by reacting with thecarbonyl moiety of the early glycosylation product that is formed by theinitial glycosylation of the protein.

It is the carbonyl group located near the junction between sugar andprotein segments of the early glycosylation product that is theorized tocomprise an active site that causes the further cross-linking of theprotein to form the advanced glycosylation end product, and likewisecontributes to the entrapment of other proteins that is evident in thedevelopment in vivo of conditions such as skin wrinkling, certain kidneydiseases, atherosclerosis, osteoarthritis and the like. Similarly, plantmaterial that undergoes nonenzymatic browning deteriorates and, in thecase of foodstuffs, become spoiled and inedible. Thus, the reaction ofthe compounds of this invention with this carbonyl moiety is believed toinhibit the late stage Maillard effect.

The rationale of the invention is to use agents which block thepost-glycosylation step, i.e., the formation of fluorescent chromophoressuch as that identified in Pongor, et al., supra. whose presence isassociated with, and leads to, the adverse sequelae of diabetes andaging. An ideal agent would prevent the formation of the chromophore andits associate cross-links of proteins to proteins and trapping ofproteins on the other proteins, such as occurs in arteries and in thekidney.

Accordingly, the compositions useful in the present invention compriseor contain agents capable of reacting with the active carbonylintermediate of the early glycosylation product. Suitable agents are thehydrazine derivatives which bear an electron-withdrawing group of thepresent invention. These agents possess an active nitrogen-containingsubstituent that is believed to react with the carbonyl of the earlyglycosylation product. Consequently, reaction of the agents with theglycosyl-lysine moiety of a protein would prevent this moiety fromforming crosslinks with other groups. Hollis and Strickberger(Diabetologia 28: 282-5 [1985]) found that in vivo administration of thecompound alpha-hydrazinohistidine, a known inhibitor of the enzymehistidine decarboxylase, reduces the accumulation of albumin in theaortas of rats. The authors proposed that the drug acted to reduceproduction of histamine in this tissue, and that histamine is thereforethe mediator of low density lipoprotein accumulation which is implicatedin atherosclerotic disease. The findings of Hollis and Strickberger aredistinguishable from the concept and application of the presentinvention on several grounds. The mechanism of histamine synthesissuppression by alpha-hydrazinohistidine suggested by the authors, isfunctionally distinct from the underlying concept of the presentinvention, and it is believed, may even be placed in question by thelatter. Additionally, one should note that alpha-hydrazinohistidine hasno electron-withdrawing group attached to the hydrazine moiety and wouldnot be expected to react efficiently and irreversibly with aglycosylation product.

Thus, the agents of the present invention have been identified andtested on the basis of their ability to react with the carbonyl moietyof the early glycosylation product to form a highly stable adduct, andwould not have been suggested from the work of Hollis and Strickberger.In particular, amino-guanidine is known to increase levels of histamine(See Lindberg and Tornqvist, "The Inhibitory Effect of Aminoguanidine onHistamine Catabolism in Human Pregnancy", Acta Obstet. Gynecol. Scand.,45: 131-139, (1966) and alpha-hydrazinohistidine and aminoguanidinetherefore have opposing effects on histamine levels. It can therefore byseen that the present findings that both alpha-hydrazinohistidine andaminoguanidine have efficacy in vivo and in vitro to reduce proteincross-linking rules out from consideration and consequentlydistinguishes the mechanism proposed by Hollis and Strickberger as theexplanation of the manner in which the compounds of the presentinvention might work to reduce advanced glycosylation end productformation.

In the instance where the composition of the present invention isutilized for in vivo or therapeutic purposes, it may be noted that thecompounds or agents used therein are biocompatible. Pharmaceuticalcompositions may be prepared with a pharmaceutically effective quantityof the agents or compounds of the present invention and may include apharmaceutically acceptible carrier, selected from known materialsutilized for this purpose. Such compositions may be prepared in avariety of forms, depending on the method of administration. Forexample, a compound may be converted to the hydrochloride salt from thecommercially available bicarbonate salt to improve its solubility and tomake it less irritating for intraperitoneal injection. Various otherpharmaceutically acceptable acid addition salts of the compounds offormulae I and II may likewise be utilized. Such acid addition salts maybe derived from a variety of organic and inorganic acids such assulfuric, phosphoric, hydrochloric, hydrobromic, hydroiodic, sulfamic,citric, lactic, maleic, succinic, tartaric, cinnamic, acetic, benzoic,gluconic, ascorbic and related acids. Also, a liquid form would beutilized in the instance where administration is by intravenous orintraperitoneal injection, while if appropriate, tablets, capsules,etc., may be prepared for oral administration. For topical applicationto the skin or eye, a solution, lotion or ointment may be formulatedwith the agent in a suitable vehicle, perhaps including a carrier to aidin penetration into the skin or eye. Other suitable forms foradministration to other body tissues are also contemplated.

The present invention likewise relates to methods for inhibiting theformation of advanced glycosylation end products, which comprisecontacting the target proteins with the composition of the presentinvention. In the instance where the target proteins are contained infoodstuffs, whether plant of animal origin, these foodstuffs could haveapplied to them by various conventional means a composition containingthe present agents. Likwise, in the instance where therapeuticapplications are intended, the animals to be treated would haveadministered to them a regular quantity of the pharmaceuticalcomposition of the present invention. Administration could take placefor example daily, and an effective quantity of the agent or compound ofthe present invention could range up to 25 mg/kg of body weight of theanimal. A topical preparation may, for example, include up to 10% of theagent or composition in an ointment or lotion for application to theskin. Naturally, some variation in these amounts is possible, and thesuggested amounts are provided in fulfillment of applicants' duty todisclose the best mode for the practice of the present invention.

As is apparent from a discussion of the environment of the presentinvention, the present methods and compositions hold the promise forarresting the aging of key proteins both in animals and plants, andconcomitantly, conferring both economic and medical benefits as a resultthereof. In the instance of foodstuffs, the administration of thepresent composition holds the promise for retarding food spoilagethereby making foodstuffs of increased shelf life and greateravailability to consumers. Replacement of currently-used preservatives,such as sulfur dioxide known to cause allergies and asthma in humans,with non-toxic, biocompatible is a further advantage of the presentinvention.

The therapeutic implications of the present invention relate to thearrest of the aging process which has as indicated earlier, beenidentified in the aging of key proteins by advanced glycosylation andcross-linking. Thus, body proteins such as collagen, elastin, lensproteins, nerve proteins and kidney glomerular basement membranes wouldall benefit in their longevity and operation from the practice of thepresent invention. It is further theorized that the present inventionwould reduce the incidence of pathologies involving the entrapment ofproteins by cross-linked target proteins, such as atherosclerosis,osteoarthritis, periarticular rigidity, loss of elasticity and wrinkingof skin, stiffening of joints, glomerulnephritis, etc. Likewise, all ofthese conditions are in evidence in patients afflicted with diabetesmellitus. Thus, the present therapeutic method is relevant to treatmentof the noted conditions in patients either of advanced age or thosesuffering from one of the mentioned pathologies.

The aminoguanidine derivatives encompassed in formula I are convenientlyprepared by chemical syntheses well known in the art. Certain of thecompounds encompassed by formula I are known compounds readily availablefrom chemical supply houses and/or preferably by synthetic methodsspecifically published therefor. The novel compounds of formula II areprepared by analogous routes. For instance, 1,3-diaminoguanidinemonohydrochloride and 2-hyrazino-2-imidazoline hydrobromide areavailable from Aldrich Chemical Company. Acetic acid hydrazide andL-glutamic acid-gamma-hydrazine hydrate can be obtained from SigmaChecmical Company. Methanesulfonyl hydrazide is obtainable fromLancaster Chemical Co. N-hydroxyhydrazinecarboximidamide tosylate can besynthesized according to the procedure of J. Med. Chem., 27, 236-238(1984). Likewise, the procedure describing1-methylhydrazinecarboximidamide tosylate is published in J. Med. Chem.,25, 505-518 (1982). N-(3-dimethylaminopropyl)hydrazinecarboximidamidedihydrobromide hydrate is mentioned in U.S. Pat. No. 4,544,759 (1985).

Other compounds described in the chemical and patent literature andencompassed by formula I are:

N-methylhydrazinecarboximidamide;

N-ethylhydrazinecarboximidamide;

N-propylhydrazinecarboximidamide;

N-butylhydrazinecarboximidamide;

N-hexylhydrazinecarboximidamide;

N,N'-dimethylhydrazinecarboximidamide;

N,N'-diethylhydrazinecarboximidamide;

N,N'-diisopropylhydrazinecarboximidamide;

N-(3-diethylaminopropyl)hydrazinecarboximidamide;

N-(2-diethylaminoethyl)hydrazinecarboximidamide;

N-(2-dimethylaminoethyl)hydrazinecarboximidamide;

N-[2-(4-methylpiperazinyl)ethyl]hydrazinecarboximidamide;

N-[2-(1-pyrrolidinyl)ethyl]hydrazinecarboximidamide;

N-[2-(1-piperidinyl)ethyl]hydrazinecarboximidamide;

N-[2-(1-hexahydroazepinyl)ethyl]hydrazinecarboximidamide;

N-[2-(4-methyl-1-hexahydro-1,4-diazepinyl)propyl]hydrazinecarboximidamide;

N-[2-(1-hexahydroazocinyl)ethyl]hydrazinecarboximidamide;

N-[2-(1-octahydroazoninyl)ethyl]hydrazinecarboximidamide; and

N-[2-(2,4-dimethyl-1-pyrrolidinyl)ethyl]hydrazinecarboximidamide.

EXAMPLE I

The following methods were used to evaluate the compounds of the presentinvention for their ability to prevent the glucose-mediatedcross-linking of protein in vitro. The test protein utilized is bovineserum albumin (BSA) at a concentration of 100 milligrams per milliliterin a 0.5M sodium phosphate buffer at pH 7.4. Glucose is included in thereaction mixture at a concentration of 200 mM. Sodium azide, 3 mM, isincluded in all solutions to prevent the growth of microorganisms.

To evaluate compounds, they are included in the above reaction mixtureat either 1 mM, 10 mM, or 100 mM. An additional set of incubationmixtures also is prepared in the absence of glucose to serve as baselinecontrols for each inhibitor. A BSA plus glucose mixture in the absenceof any inhibitor serves as an indication of the maximum amount ofcross-linking that can occur in each mixture.

After incubation of the mixtures for three weeks at 37° C., the BSA ineach mixture must be isolated from the other components of the mixturebefore the degrees of browning is determined. This is necessary becausemany of the inhibitors are either fluorescent themselves or quench thefluorescence of the browned BSA. To effect the separation, the BSA isprecipitated by the addition of 1.0 milliliters of saturated ammoniumsulfate to each 100 microliters of incubation mixture. The resultingprecipitate is centrifuged and the supernatant solutions are discarded.The precipitate is washed once with saturated ammonium sulfate, then theBSA pellet is redissolved in 1 milliliter of phosphate-buffered saline(PBS) to give a final protein concentration of about 10 milligrams permilliliter.

The actual protein concentration of the BSA solution is determined by astandard dye-binding protein assay. The fluorescence of the BSA ismeasured in a spectrofluorimeter at an excitation wavelength of 370nanometers and an emission wavelength of 440 nanometers. Thiscorresponds to the detection of chromophores including FFI which haveformed in the BSA as a formation of advanced glycosylation endproductsthrough the reaction of glycosylated amino groups.

The specific fluorescence of the BSA is measured as fluorescence (inarbitrary units) per milligram of BSA. It is expressed as the increasein fluorescence during the incubation period of the sample incubatedwith glucose minus the corresponding value in the absence of glucose.The degree of inhibition of each compound is expressed in a percentagescale, where 0% represents no inhibition of browning, i.e., thefluorescence developed in an incubation mixture containing only glucoseand BSA, in the absence of any inhibitors. One hundred percentinhibition corresponds to the degree of fluorescence developed in theabsence of glucose.

Following the above procedure, the following results were obtained usingthe test compounds at a concentration of 10 mM.

Percent inhibition of browning by various compounds at 10 mM:

    ______________________________________                                        85%  1,2,3-triaminoguanidine hydrochloride                                    84%  1,3-diaminoguanidine monohydrochloride                                   81%  N--hydroxyhydrazinecarboximidamide tosylate                              76%  2-hydrazino-2-imidazoline hydrobromide                                   65%  L-glutamic acid-gamma-hydrazide hydrate                                  63%  N,N"--3,3'-[1,4-piperazinediylbis(3,1-                                        propanediyl)]bishyrazinecarboximidamide                                       tetrahydrobromide                                                        59%  N--(3-dimethylaminopropyl)hydrazinecarboximidamide                            dihydrobromide hydrate                                                   59%  N--(3-(4-methylpiperazin-1-yl)propyl)-                                        hydrazinecarboximidamide trihydrobromide                                 53%  1-methylhydrazinecarboximidamide tosylate                                49%  methanesulfonyl hydrazide                                                48%  acetic acid hydrazide                                                    45%  1-(2-hydroxyethyl)hydrazinecarboximidamide sulfate                            2:1                                                                      42%  N--(2,2-dimethyl-3-dimethylaminopropyl)hydrazine-                             carboximidamide dihydrobromide                                           41%  N--(3-(4-morpholino)propyl)hydrazinecarboximidamide                           dihydrobromide                                                           40%  aminoguanidine hydrochloride                                              0%  no inhibitor                                                             ______________________________________                                    

EXAMPLE 2

Evaluation of test compounds at 1 mM was performed the same way as inExample 1. The results are as follows:

    ______________________________________                                        42%  2-hydrazino-2-imidazoline hydrobromide                                   37%  N--hydroxyhydrazinecarboximidamide tosylate                              37%  1,2,3-triaminoguanidine hydrochloride                                    37%  N,N"--3,3'-[1,4-piperazinediylbis(3,1-                                        propanediyl)]bishydrazinecarboximidamide                                      tetrahydrobromide                                                        33%  1,2-diamino-2-imidazoline tosylate                                       31%  1,3-diaminoguanidine monohydrochloride                                   26%  acetic acid hydrazide                                                    24%  L-glutamic acid-gamma-hydrazide hydrate                                  23%  N--(3-dimethylaminopropyl)hydrazinecarboximidamide                            dihydrobromide hydrate                                                   23%  N--(3-(4-methylpiperazin-1-yl)propyl)-                                        hydrazinecarboximidamide trihydrobromide                                 22%  beta-aspartyl hydrazide                                                  21%  1-(2-hydroxyethyl)hydrazinecarboximidamide sulfate                            2:1                                                                      21%  methanesulfonyl hydrazide                                                19%  aminoguanidine hydrochloride                                             ______________________________________                                    

EXAMPLE 3

Evaluation of test compounds at 100 mM was performed the same way as inExample 1. The results are as follows:

Percent inhibition of browning by various compounds at 100 mM.

    ______________________________________                                        100% N,N"--3,3'-[1,4-piperazinediylbis(3,1-                                        propanediyl)]bishydrazinecarboximidamide                                      tetrahydrobromide                                                        98%  L-glutamic acid-gamma-hydrazide hydrate                                  98%  1,3-diaminoguanidine monohydrochloride                                   97%  N--(2,2-dimethyl-3-dimethylaminopropyl)hydrazine-                             carboximidamide dihydrobromide                                           96%  N--hydroxyhydrazinecarboximidamide tosylate                              96%  N--(3-(4-methylpiperazin-l-yl)propyl)-                                        hydrazinecarboximidamide trihydrobromide                                 95%  2-hydrazino-2-imidazoline hydrobromide                                   93%  methanesulfonyl hydrazide                                                93%  N--(3-(4-morpholino)propyl)hydrazinecarboximidamide                           dihydrobromide                                                           92%  N--(3-dimethylaminopropyl)hydrazinecarboximidamide                            dihydrobromide hydrate                                                   88%  aminoguanidine hydrochloride                                             ______________________________________                                    

The in vitro experiments of Examples 1-3 indicate that this type of drugtherapy has benefit in reducing the pathology associated with theadvanced glycosylation of proteins and the formation of crosslinksbetween proteins and other macromolecules. Drug therapy may be used toprevent the increased trapping and cross-linking of proteins that occursin diabetes and aging which leads to sequelae such as retinal damage,and extra-vascularly, damage to tendons, ligaments and other joints.This therapy might retard atherosclerosis and connective tissue changesthat occur with diabetes and aging. Both topical, oral, and parenteralroutes of administration to provide therapy locally and systemically arecontemplated.

EXAMPLE 4

Certain of the novel aminoguanidine derivatives are synthesized in thefollowing manner.

N-(3-(4-morpholino)propyl)hydrazinecarboximidamide dihydrobromide

Hydrazinecarboximidothioic acid ethyl ester hydrobromide (10.0 grams)and 3-(4-morpholino)propylamine (7.56 grams) are dissolved in ethanol(20 Milliliters) and kept at room temperature for 2 days, then heated atreflux for 30 minutes. Isopropanol (20 milliliters) is added, and themixture is cooled and treated with 48% hydrobromic acid (6 milliliters).Additional ethanol (50 milliliters) and isopropanol (20 milliliters) areadded and the mixture is stored at -20° C. for two days. The crystallineprecipitate is triturated, filtered out and washed with ethanol andisopropanol, giving 14.91 grams of crystalline solid. To purifying thismaterial, 11 grams are dissolved in 16.5 milliliters of water, filteredto remove insoluble material, and diluted with 5.5 milliliters ofmethanol and 100 milliliters of isopropanol. After storage at roomtemperature and at 4° C., the precipitate is filtered out and washedwith isopropanol, giving 9.0 grams of colorless crystals of the titlecompound, melting point of 129°-130° C.

Following analogous procedurs, the following aminoalkylhydrazinecarboximadimide derivatives are prepared (substituting for3-(4-morpholino)propylamine the following reagents):

From 3-(4-methylpiperazin-1-yl)propylamine, the compoundN-(3-(4-methyl-piperidin-1-yl)propyl)hydrazinecarboximidamidetrihydrobromide, melting point 212° C.

From 2,2-dimethyl-3-dimethylaminopropylamine, the compoundN-(2,2-dimethyl-3-dimethylaminopropyl)hydrazinecarboximidamidedihydrobromide, melting point 105°-107° C.

From 1,4-piperazinediylbis(3,1-propylamine), the compoundN,N"-3,3'-[4-piperazinediylbis(3,I-propanedyl)]bishydrazinecarboximidamide tetrahydrobromide, melting point 241°-244°C.

From 3-dimethylaminopropylamine, the compoundN-(3-dimethylaminopropyl)hydrazinecarboximidamide dihydrobromide,melting point 82°-84° C.

EXAMPLE 5 1-(2-hydroxyethyl)hydrazinecarboximidamide sulfate 2:1

Carbimidothioic acid methyl ester sulfate 2:1 (6.955 grams) and2-hydroxyethylhydrazine (9.13 grams) are stirred and heated at 40° C.for one hour. Methanol (20 milliliters) is added and the mixture isheated at reflux for four hours. On cooling, crystals separate.Filtration gives 5.34 grams of colorless crystals. Threerecrystallization from 88% methanol afford 4.111 grams of the titlecompound as colorless crystals, melting point 178.5°-180° C.

EXAMPLE 6 2-(1-(2-hydroxyethyl)hydrazino)-1-imidazoline sulfate (2:1)

2-Methylthio-2-imidazoline sulfate (2:1) (1.98 g) and2-hydroxyethylhydrazine (2.12 g) are heated in ethanol (3 ml) at refluxfor 1 hr., then stirred at 25° C. for 3 hr. The solution was dilutedwith ethanol (20 ml) and kept at 4° C. for 18 hr. The crystals whichseparated were filtered out and washed with ethanol. Weight 811 mg, mp190°-4° C.

Similarly, from the corresponding S-methylisothiuronium derivatives areprepared the following compounds or their acid addition salts:

2-(1-(2-hydroxyethyl)hydrazino)-3-methyl-1-imidazoline;

2-(1-(2-hydroxyethyl)hydrazino)-4,4-dimethyl-1-imidazoline;

1,3-dimethyl-2-imidazolidinone 1-(2-hydroxyethyl)hydrazone;

2-(1-(2-hydroxyethyl)hydrazino)-3,4,5,6-tetrahydropyrimidine;

2-(1-(2-hydroxyethyl)hydrazino)-5,5-dimethyl-3,4,5,6-tetrahydropyrimidine;

2-(1-(2-hydroxyethyl)hydrazino)-5-hydroxy-3,4,5,6-tetrahydropyrimidine;

2-(1-(2-hydroxyethyl)hydrazino)-5,5-dibutyl-3,4,5,6-tetrahydropyrimidine;

2-(1-(2-hydroxyethyl)hydrazino)-3-methyl-3,4,5,6-tetrahydropyrimidine;

1,3-dimethyl-3,4,5,6-hexahydro-2(1H)-pyrimidone1-(2-hydroxyethyl)hydrazone;

2-(1-(2-hydroxyethyl)hydrazino)-4,5,6,7-tetrahydro-1,3(1H)-diazepine;

2-(1-(2-hydroxyethyl)hydrazino)-4,4,7,7-tetramethyl-4,5,6,7-tetrahydro-1,3(1H)-diazepine;

N-methyl-1-(2-hydroxyethyl)hydrazinecarboximidamide;

N,N'dimethyl-1-(2-hydroxyethyl)hydrazinecarboximidamide;

1-pyrrolidinecarboximidic acid 1-(2-hydroxyethyl)hydrazide;

N-methyl(1-pyrrolidine)carboximidic acid 1-(2-hydroxyethyl)hydrazide;

1-piperidinecarboximidic acid 1-(2-hydroxyethyl)hydrazide;

1-hexahydroazepinecarboximidic acid 1-(2-hydroxyethyl) hydrazide;

1-(4-methylpiperazine)carboximidic acid 1-(2-hydroxyethyl) hydrazide;

1-(4-methylhexahydro-1,4-diazepine)carboximidic acid1-(2-hydroxyethyl)hydrazine; and

4-morpholinecarboximidic acid 1-(2-hydroxyethyl)hydrazide.

EXAMPLE 7 1,2-Diamino-2-imidazoline p-toluenesulfonate

1-Aminoimidazolidine-2-thione (2.34 g) and methyl p-toluenesulfonate(4.1 g) in ethanol (15 ml) are heated to reflux for 10 minutes, thenkept at room temperature for 16 hours. The crystalline precipitate isfiltered out and washed with isopropanol to give 4.613 g of1-amino-2-methylthio-2-imidazoline p-toluenesulfonate as colorlessneedles, of which 3.79 g is placed in methanol (15 ml) and treated withconcentrated aqueous ammonia. After stirring for 6 hours, the mixture isdiluted with 20 ml isopropanol. After another 12 hours, 5 ml of liquidis distilled off at atmospheric pressure, and 10 ml isopropanol is addedto the remainder. The crystals which separate on cooling are filteredout and washed with isopropanol to give 2.323 g of1,2-diamino-2-imidazoline p-toluenesulfonate, melting point 190°-190.5°C.

EXAMPLE 8

The aminoguanidine derivatives of the present invention are testedaccording to the method of Stoner, Agents and Actions, 17, pp. 5-9(1985) in order to ascertain their lack of ability to inhibit the enzymediamine oxidase. This enzyme is responsible for detoxifying histamineand therefore it would be desirable in any therapy to avoid inhibitionof this enzyme.

Percent Inhibition at 10 micromolar:

    ______________________________________                                        92%  Aminoguanidine HCl                                                       0%   1-(2-hydroxyethyl)hydrazinecarboximidamide sulfate                            2:1                                                                      0%   1-methylhydrazinecarboximidamide tosylate                                84%  N--hydroxyhydrazinecarboximidamide tosylate                              59%  2-hydrazino-2-imidazoline hydrobromide                                   92%  1,3-diaminoguanidine monohydrochloride                                   0%   N--(3-dimethylaminopropyl)hydrazinecarboximidamide                            dihydrobromide hydrate                                                   0%   N--(3-(4-methylpiperazin-l-yl)propyl)-                                        hydrazinecarboximidamide trihydrobromide                                 12%  N,N"--3,3'-[1,4-piperazinediylbis(3,1-                                        propanediyl)]bishydrazinecarboximidamide                                      tetrahydrobromide                                                        82%  1,2,3-triaminoguanidine hydrochloride                                    5%   N--(3-(4-morpholino)propyl)hydrazinecarboximidamide                           dihydrobromide                                                           0%   N--(2,2-dimethyl-3-dimethylaminopropyl)hydrazine-                             carboximidamide dihydrobromide                                           9%   1,2-diamino-2-imidazoline tosylate                                       9%   methanesulfonyl hydrazide                                                75%  L-glutamic acid-gamma-hydrazide hydrate                                  32%  beta-aspartyl hydrazide                                                  0%   acetic acid hydrazide                                                    ______________________________________                                    

This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allrespects illustrative and not restrictive, the scope of the inventionbeing indicated by the appended Claims, and all changes which comewithin the meaning and range of equivalency are intended to be embracedtherein.

What is claimed is:
 1. The compounds of the formula ##STR6## wherein R₈,R₉ and R₁₀ are hydrogen or a lower alkyl group, and m is an integer of2-4, and their pharmaceutically acceptable acid addition salts.
 2. Thecompounds according to claim 1 wherein R₈, R₉ and R₁₀ are each hydrogen.3. The compounds according to claim 1 wherein m is
 2. 4. The compoundaccording to claim 1 which is 1,2-diamino-2-imidazolinep-toluenesulfonate.